Abstract
Single-molecule Förster resonance energy transfer (smFRET) has emerged as a powerful tool for elucidating biological structure and mechanisms on the molecular level. Here, we focus on applications of smFRET to study interactions between DNA and enzymes such as DNA and RNA polymerases. SmFRET, used as a nanoscopic ruler, allows for the detection and precise characterisation of dynamic and rarely occurring events, which are otherwise averaged out in ensemble-based experiments. In this review, we will highlight some recent developments that provide new means of studying complex biological systems either by combining smFRET with force-based techniques or by using data obtained from smFRET experiments as constrains for computer-aided modelling.
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Abbreviations
- AFM:
-
Atomic force microscopy
- ALEX:
-
Alternating-laser excitation
- ATP:
-
Adenosine tri-phosphate
- BVA:
-
Burst variance analysis
- bp:
-
Base pair
- CAP:
-
Catabolite activator protein
- DNA:
-
Deoxyribonucleic acid
- dsDNA:
-
Double-stranded deoxyribonucleic acid
- dNTP:
-
Deoxyribonucleoside tri-phosphate
- FRET:
-
Förster resonance energy transfer
- FPS:
-
FRET-restrained positioning and screening
- MFD:
-
Multiparameter fluorescence detection
- NMR:
-
Nuclear magnetic resonance
- NPS:
-
Nano-positioning system
- PDA:
-
Probability distribution analysis
- PIFE:
-
Protein-induced fluorescence enhancement
- Pol:
-
Polymerase
- quFRET:
-
Quenchable Förster resonance energy transfer
- RNA:
-
Ribonucleic acid
- RNAP:
-
RNA polymerase
- rNTP:
-
Ribonucleoside tri-phosphate
- smFRET:
-
Single-molecule Förster resonance energy transfer
- SSB:
-
Single-stranded DNA binding protein
- ssDNA:
-
Single-stranded deoxyribonucleic acid
- TIRF:
-
Total internal reflection fluorescence
- TBP:
-
TATA box binding protein
- TF:
-
Transcription factor
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Acknowledgments
We thank Herbert van Amerongen and Arjen Bader for critically reading the manuscript and providing helpful suggestions. S.F. acknowledges funding from the Foundation for Fundamental Research on Matter (FOM), which is part of the Netherlands Organisation for Scientific Research (NWO).
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The authors declare that they have no conflict of interest.
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S. Farooq and C. Fijen contributed equally to this study.
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Farooq, S., Fijen, C. & Hohlbein, J. Studying DNA–protein interactions with single-molecule Förster resonance energy transfer. Protoplasma 251, 317–332 (2014). https://doi.org/10.1007/s00709-013-0596-6
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DOI: https://doi.org/10.1007/s00709-013-0596-6